Synthesis of novel xylose free occidiofungin analogues and methods of using them

ABSTRACT

The subject invention pertains to methods for producing novel occidiofungin analogues lacking xylose (OF-Δxyl analogue). OF-Δxyl analogues can contain aldehyde, amine, triazole, or hydrazones. The aldehyde, amine, triazole or hydrazone containing analogues of OF-Δxyl can be further modified by substitution with additional functional groups. Various OF-Δxyl analogues described herein have reduced inflammation and toxicity and superior pharmacological properties compared to the natural occidiofungins. Accordingly, certain embodiments of the invention provide methods of treating or preventing fungal infections by administering to the subjects in need thereof the OF-Δxyl analogues described herein.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Ser.No. 62/653,253, filed Apr. 5, 2018, the disclosure of which is herebyincorporated by reference in its entirety, including all figures, tablesand amino acid or nucleic acid sequences.

This invention was made with government support under 1R41A1122441-01A1awarded by the NIH-NIAID. The government has certain rights in theinvention.

The Sequence Listing for this application is labeled “Seq-List.txt”which was created on Apr. 5, 2019 and is 1 KB. The entire content of thesequence listing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Fungal infections caused by pathogens that are resistant to commonlyused classes of antifungals are becoming increasingly prevalent. A CDCreport described the spread of multi-drug resistant Candida auriscausing systemic infections in hospitalized patients. Furthermore, otherspecies such as Candida glabrata and Candida parapsilosis have beenreported to have gained resistance to routinely used azoles andechinocandins. An example of a fungal infection that is rapidlydeveloping resistance to currently available treatments is vulvovaginalcandidiasis (VVC). VVC affects approximately 75% of women and 5-10% ofwomen develop recurrent VVC (RVVC). Approximately 90% of VVC is causedby C. albicans, while the remaining 10% is caused by C. glabrata,Candida tropicalis, Candida parapsilosis and Candida krusei. Thesenon-albicans VVC causing species are generally resistant to azoletreatments, which are the most common treatment option for VVC. No newtherapeutic developments occurred in decades for RVVC. There is noestablished treatment method for VVC, which has led to the developmentof several ineffective treatment methods. These generally include theuse of a rigorous dosing regimen of antifungals followed by a longperiod of prophylactic dosing.

Candida species are an important cause of infection and mortality in allhospitalized patients. Candidemia has a mortality rate of 30%-50% incancer patients and is a major complicating factor in the treatment ofcancer. Despite current antifungal drugs, invasive fungal infections arestill a major cause of morbidity and mortality in transplant patients.Reports suggest that candidal infection is the first and second mostcommon infection in lung and heart transplant recipients, respectively.In heart transplant recipients, candidal disease has been attributed toa mortality rate of 28%. A rise in candidemia caused by non-albicansCandida spp. and an increase in azole resistance is alarming; andsupports the need for new antifungals. More appropriate antifungaltreatment options may reduce the cost of treatment and mortality ofpatients.

Antifungals currently in clinical use primarily comprise of thepolyenes, echinocandins and azoles. The polyene antifungal amphotericinB was introduced in the 1950s and was the only antifungal availableuntil the introduction of the azole class of antifungals in the 1980s.These two groups primarily target ergosterol production or bind toergosterol, disrupting the fungal membrane. The echinocandins, the thirdgroup, are synthetically modified lipopeptides that originate from anatural cyclic peptide produced by fungi. This group selectivelyinhibits 1,3-β-glucan synthesis by functioning as a non-competitiveinhibitor of 1,3-β-glucan synthase. Widespread resistance and theineffective spectrum of activity of these antifungals have beenreported. The prevalence of echinocandin and azole-resistant fungalpathogens and the limited spectrum of activity of those compounds is onemajor issue contributing to the need for a new class of antifungals.Additionally, current antifungal treatments lead to abnormal liver andkidney function tests and have limitations with respect to theirspectrum of activity and toxicities. Presumably, the identification of anovel class of antifungals with a broad spectrum of activity and aunique mechanism of action would mitigate the loss of life associatedwith the use of the current classes of antifungals. These limitationsand toxicity problems have created an urgent need to identify antifungalcompounds that have new fungal cell targets.

Occidiofungins, isolated from Burkholderia contaminans MS14, are a newlydiscovered class of antifungals. Occidiofungin is a non-ribosomallysynthesized glycolipopeptide produced by B. contaminans MS14, a soilbacterium. It is a cyclic peptide with a base mass of 1200 Da (FIG. 1).The bacterium produces a mixture of structural analogues of the basecompound (Occidiofungins A-D). However, all analogues are composed ofeight amino acids and a C18 fatty amino acid (hereinafter, NAA)containing a xylose sugar, and a 2,4-diaminobutyric acid (DABA). Thestructural analogues differ by the addition of oxygen to asparagine 1(Asn1) forming a β-hydroxy asparagine 1 (BHN1) and by the addition ofchlorine to β-hydroxy tyrosine 4 (BHY) forming 3-chloro β-hydroxytyrosine 4 (chloro-BHY). Occidiofungins (A-D) show promise for thedevelopment of novel therapeutics for treating fungal infections.

Improvements in the production of the occidiofungin B analog have beenmade. The method devised does not lead to a complex mixture ofoccidiofungin.

Mammalian glycoproteins containing xylose are not reported. Xylose isone of the main cross-reactive carbohydrate determinants (CCDs) thatlead to an allergic response. An allergic or stress response wasobserved in mice following an intravenous administration ofoccidiofungin (A-D). Therefore, producing xylose free occidiofungin(OF-Δxyl) analogues and determining their efficacy against fungalinfections is desirable.

BRIEF SUMMARY OF THE INVENTION

The invention provides methods for producing novel occidiofunginanalogues that lack xylose (OF-Δxyl analogues) and methods of usingOF-Δxyl analogues. In certain embodiments, OF-Δxyl analogues arealdehyde containing analogues. In certain embodiments, the aldehydecontaining OF-Δxyl analogues are further modified to produce aminecontaining OF-Δxyl analogues, triazole containing OF-Δxyl analogues orhydrazones containing OF-Δxyl analogues. The aldehyde, amine, triazoleor hydrazone containing OF-Δxyl analogues can be further modified toproduce additional OF-Δxyl analogues. Various OF-Δxyl analoguesdescribed herein induce reduced inflammation and toxicity and havesuperior pharmacological properties compared to the naturaloccidiofungins. Accordingly, certain embodiments of the inventionprovide methods of controlling fungal infections by administering to thesubjects in need thereof the OF-Δxyl analogues described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Structure of occidiofungin. The location of the diol is atcarbons 5 and 6 (as numbered in the Figure).

FIGS. 2A-2D. A, B, C and D provide the structures of aldehyde, amine,triazole and hydrazone containing OF-Δxyl analogues, respectively.

FIGS. 3A-3E. A provides a scheme of oxidative cleavage for the synthesisof aldehyde containing OF-Δxyl analogues, B describes reductiveamination for the synthesis of amine containing OF-Δxyl analogues, Cprovides a scheme for the synthesis of triazole containing OF-Δxylanalogues and D describes a scheme for the synthesis of hydrazonecontaining OF-Δxyl analogues, E describes a scheme for synthesis ofthree acyl lipid analogs.

FIGS. 4A-4B. Generation and characterization of the aldehyde analog ofoccidiofungin. (A) HPLC isolation of the aldehyde analog ofoccidiofungin. The solvent used was 20% methanol in water. The blackline represents the absorbance unit (AU) at 220 nm. (B) ESI-MSchromatograms of the aldehyde analog (868 Da) of occidiofungin. The datashows that the oxidative cleavage went to completion, given that thereis no mass of starting material (1216 Da).

FIGS. 5A-5D. HPLC chromatograph of 100 μg of wild-type occidiofungin(A), aldehyde containing OF-Δxyl analogues following the reaction withundecylamine (B), dodecylamine (C), and DL-dihydrosphingosine D). Thepeak indicated by arrow is a desired product. Grey lines represent thegradient of buffer B (water with 0.1% TFA), while black lines representthe absorbance unit (AU) at 220 nm.

FIGS. 6A-6E. ESI-MS of OF-Δxyl analogues. (A) wild-type, (B) an aldehydecontaining OF-Δxyl analogue, (C) an aldehyde containing OF-Δxyl analoguefollowing a reaction with undecylamine, (D) an aldehyde containingOF-Δxyl analogue following a reaction with dodecylamine, and (E) analdehyde containing OF-Δxyl analogue following a reaction withDL-dihydrosphingosine.

FIG. 7. Efficacy of occidiofungin in treating murine vulvovaginalcandidiasis. The graph demonstrates CFUs per ml of Candida albicans inthe control group of mice compared to the groups treated intravaginallywith different concentrations of occidiofungin in 0.3% noble agar. Errorbars represent standard deviation. Statistical analyses indicate asignificant difference between the control group and the treated groups(p<0.001) as indicated by the asterisk.

FIGS. 8A-8B. TOCSY and NOESY NMR spectra of occidiofungin. (A) TOCSYspin system correlations of the aldehyde occidiofungin product.Fingerprint region (NH correlations), alpha to side chain correlationsand side chain correlations are shown. Abbreviations are: diaminobutyric acid 5 (DABAS), novel amino acid 2 (NAA2),beta-hydroxy-asparagine 1 (BHN1), and beta-hydroxy-tyrosine 4 (BHY4).(B) NOESY spin system correlations. The expansion shows theintra-residue NOE interaction of the aldehyde proton of NAA2 to theamide proton of the NAA2.

FIGS. 9A-9D. Co-sedimentation assay demonstrating the binding ofoccidiofungin analogs to actin. (A) Binding curve of nativeoccidiofungin to actin (Kd=1000 nM; the stoichiometry [ligand:protein]is 25: 1. (B) Binding curve of the dodecylamine analog to actin (Kd=4200nM; the stoichiometry [ligand:protein] is 34: 1. (C) Binding curve ofphalloidin to actin (Kd=8 nM; the stoichiometry [ligand:protein] is0.7:1. (D) Binding curve of the dihydrosphingosine analog to actin(Kd=25 nM; the stoichiometry [ligand:protein] is 1.8:1. The graph isplotted between the amount of free compound obtained in the supernatantof the co-sedimentation assay and the amount of bound compound obtainedfrom the actin pellet. Data for native occidiofungin and phalloidin hasbeen published previously (Ravichandran et al.).

FIGS. 10A-10B. Pyrene labeled actin polymerization and depolymerizationassay. The effect of phalloidin, native occidiofungin, and thedihydrosphingosine analog on actin (A) polymerization and (B)depolymerization assays. (grey •) represents buffer baseline control;(▪) represents no drug control; (♦) represents phalloidin treatment;(black •) represents native occidiofungin treatment; (▴) representsdihydrosphingosine analog treatment. The fluorescent data is measured interms of arbitrary units (AU).

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1: Sequence of cyclic occidiofungin antibiotic. Asn1-[NovelAmino Acid 2 (NAA2)-Ser3-BHY4-Gly6-Asn7-Ser8].

DETAILED DISCLOSURE OF THE INVENTION

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Further, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising.”

The phrases “consisting essentially of” or “consists essentially of”indicate that the claim encompasses embodiments containing the specifiedmaterials or steps and those that do not materially affect the basic andnovel characteristic(s) of the claim.

The term “about” means within an acceptable error range for theparticular value as determined by one of ordinary skill in the art,which depend in part on how the value is measured or determined, i.e.,the limitations of the measurement system. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed. In the context of compositions containingamounts of ingredients where the terms “about” or “approximately” areused, these compositions contain the stated amount of the ingredientwith a variation (error range) of 0-10% around the value (X±10%).

In the present disclosure, ranges are stated in shorthand, so as toavoid having to set out at length and describe each and every valuewithin the range. Any appropriate value within the range can beselected, where appropriate, as the upper value, lower value, or theterminus of the range. For example, a range of 0.1-1.0 represents theterminal values of 0.1 and 1.0, as well as the intermediate values of0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate rangesencompassed within 0.1-1.0, such as 0.2-0.5, 0.2-0.8, 0.7-1.0, etc.Values having at least two significant digits within a range areenvisioned, for example, a range of 5-10 indicates all the valuesbetween 5.0 and 10.0 as well as between 5.00 and 10.00 including theterminal values.

When ranges are used herein, such as for dose ranges, combinations andsubcombinations of ranges (e.g., subranges within the disclosed range),specific embodiments therein are intended to be explicitly included.

“Pharmaceutically acceptable” means approved or approvable by aregulatory agency of the Federal or a state government or thecorresponding agency in countries other than the United States, or thatis listed in the U.S. Pharmacopoeia or other generally recognizedpharmacopoeia for use in animals, and more particularly, in humans.

“Pharmaceutically acceptable salt” refers to a salt of an occidiofunginanalogue of the invention that is pharmaceutically acceptable and thatpossesses the desired pharmacological activity of the parentoccidiofungin analogue. In particular, such salts are non-toxic may beinorganic or organic acid addition salts and base addition salts.

“Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant,excipient or carrier with which an occidiofungin analogue of theinvention is administered. A “pharmaceutically acceptable vehicle”refers to a substance that is non-toxic, biologically tolerable, andotherwise biologically suitable for administration to a subject, such asan inert substance, added to a pharmacological composition or otherwiseused to facilitate administration of an agent and that is compatibletherewith. Examples of vehicles include but are not limited to calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils, and polyethyleneglycols.

“Subject” includes humans or non-human animals, particularly, mammals,such as bovine, porcine, canine, rodent, or feline animals.

“Treating” or “treatment” of any infection refers, in one embodiment, toameliorating the infection (i.e., arresting or reducing the developmentof the disease or at least one of the clinical symptoms thereof). Inanother embodiment “treating” or “treatment” refers to ameliorating atleast one physical parameter, which may not be discernible by thesubject. In yet another embodiment, “treating” or “treatment” refers tomodulating the infection, either physically, (e.g., stabilization of adiscernible symptom), physiologically, (e.g., stabilization of aphysical parameter), or both. In yet another embodiment, “treating” or“treatment” refers to delaying the onset of the infection.

As used herein, the terms “reducing”, “inhibiting”, “blocking”,“preventing”, “alleviating”, or “relieving” when referring to anoccidiofungin analogue, mean that the occidiofungin analogue brings downthe occurrence, severity, size, volume or associated symptoms of aninfection by at least about 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%,25%, 27.5%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%,or 100% compared to how the infection would normally exist withoutapplication of the OF-Δxyl analogue or a composition comprising theOF-Δxyl analogue.

In treatment methods according to the invention, a therapeuticallyeffective amount of an OF-Δxyl analogues according to the invention isadministered to a subject suffering from or diagnosed as having such aninfection. A “therapeutically effective amount” means an amount or dosesufficient to generally bring about the desired therapeutic orprophylactic benefit in patients in need of such treatment for thedesignated infection.

Effective amounts or doses of the OF-Δxyl analogues of the presentinvention may be ascertained by routine methods such as modeling, doseescalation studies or clinical trials, and by taking into considerationroutine factors, e.g., the mode or route of administration or drugdelivery, the pharmacokinetics of the occidiofungin analogue, theseverity and course of the infection, the subject's previous or ongoingtherapy, the subject's health status and response to drugs, and thejudgment of the treating physician. An example of a dose is in the rangeof from about 0.001 to about 200 mg of an occidiofungin analogue per kgof subject's body weight per day, preferably about 0.05 to 100mg/kg/day, or about 1 to 35 mg/kg/day, even more preferably, about 1, 5,10, or 20 mg/kg/day, in single or divided dosage units (e.g., BID, TID,QID). For a 70-kg human, an illustrative range for a suitable dosageamount is from about 0.05 to about 7 g/day, preferably, about 0.07 toabout 2.45 g/day, even more preferably, about 0.07, 0.35, 0.7, or 1.4g/day.

Structure of natural analogues of occidiofungin is provided by Formula I(also provided in FIG. 1):

As seen in Formula I, natural analogues of occidiofungin are composed ofeight amino acids. One of these amino acids is NAA, which contains axylose sugar. NAA is 3 amino-5,6-dihydroxy-7-O-xylose-octadecanoic acid.The carbon atoms in NAA are numbered based on the carbonyl group of NAAas the first carbon (FIG. 1). Also, the fifth and sixth carbon atoms ofNAA each have a hydroxyl group. The seventh carbon of NAA is connectedto a xylose via an ether linkage and a C₁₁H₂₃ moiety. Certain structureand sequence information of occidiofungin is disclosed in United Statespatent application publication number 2011/0136729, which is hereinincorporated by reference in its entirety. Certain embodiments of theinstant invention provide OF-Δxyl analogues and methods of makingOF-Δxyl analogues.

In one embodiment, an OF-Δxyl analogue comprises an aldehyde group,wherein the fifth carbon of NAA is the carbonyl group of the aldehyde,while the part of NAA from the sixth carbon is removed from the parentoccidiofungin. Such OF-Δxyl analogue is referred to as aldehydecontaining OF-Δxyl analogue and it can be represented by Formula II(also provided in FIG. 2A).

In another embodiment, the carbonyl carbon of the aldehyde containingOF-Δxyl analogue is aminated to produce an analogue containing an aminegroup. Therefore, in such embodiments, the fifth carbon of NAA isconnected to an amine group while the part of NAA beyond the sixthcarbon is removed from the parent occidiofungin. Such OF-Δxyl analogueis referred to as amine containing OF-Δxyl analogue and can berepresented by Formula III (also provided in FIG. 2B).

R₄ and R₅ in an amine containing OF-Δxyl analogue can be H, OH, asubstituted or unsubstituted alkane, substituted or unsubstitutedalkene, substituted or unsubstituted alkyne, substituted orunsubstituted aryl, substituted or unsubstituted heterocycle,substituted or unsubstituted heteroaryl, substituted or unsubstitutedheterocycle, substituted or unsubstituted ether, substituted orunsubstituted thioether, substituted or unsubstituted ketone or ahalogen.

In certain embodiment, the aldehyde containing OF-Δxyl analogues can befurther modified to produce a triazole containing OF-Δxyl analogue. Insuch embodiment, the fifth carbon of NAA is connected to a triazolering, while the part of NAA from the sixth carbon and beyond is removedfrom the parent occidiofungin. Such OF-Δxyl analogue is referred to astriazole containing OF-Δxyl analogue and can be represented by FormulaIV (also provided in FIG. 2C).

In certain embodiments, the aldehyde containing OF-Δxyl analogues can befurther modified to produce a hydrazone containing OF-Δxyl analogue. Insuch embodiment, the fifth carbon of NAA is connected to a hydrazinegroup, while the part of NAA from the sixth carbon and beyond is removedfrom the parent occidiofungin. Such OF-Δxyl analogue is referred to as ahydrazone containing OF-Δxyl analogue and can be represented by FormulaV (also provided in FIG. 2D).

Certain embodiments of the invention provide methods for producing analdehyde containing OF-Δxyl analogue. Such methods comprise oxidativecleavage of NAA of a natural occidiofungin between the fifth and sixthcarbons while converting the fifth carbon to a carbonyl of the resultingaldehyde. Therefore, after an oxidative cleavage reaction, occidiofunginof Formula I is converted into an OF-Δxyl analogue of Formula II.

“Oxidative cleavage” as used herein comprises treating an occidiofunginof Formula I with appropriate reagents under appropriate conditions tocleave the carbon-carbon bond between the fifth and the sixth carbons ofNAA, and carbonyl group is formed on the fifth carbon and optionally, onthe sixth carbon.

In some embodiments, NAA is subjected to oxidative cleavage between thefifth and sixth carbons with a metaperiodate, for example, metaperiodicacid (HIO₄), sodium periodate (NaIO₄), potassium periodate (KIO₄), or apermanganate salt, such as KMnO4. In certain embodiments, oxidativecleavage between the fifth and sixth carbons with a periodate isperformed on carbohydrates in water. In specific embodiments, oxidativecleavage between the fifth and sixth carbons is performed with aperiodate in mixed aqueous organic solvents, such as acetonitrile/water,water/methanol and water/isopropanol. A skilled artisan can determine anappropriate ratio of water and the organic solvent.

Oxidative cleavage reaction with periodate converts the fifth as well asthe sixth carbons of NAA to carbonyl groups (FIG. 3A). Additionalexamples of periodates useful in such oxidative cleavage are known to askilled artisan and such embodiments are within the purview of theinvention.

In a specific embodiment, an occidiofungin of Formula I is incubatedwith NaIO₄ for about 20-40 minutes at about 60-80° C. to produce analdehyde containing OF-Δxyl analogue of Formula II (FIG. 3A). Theproduct can be further purified, for example, via column chromatography,such as HPLC.

Additional reactions designed to cleave the NAA between the fifth andsixth carbons while converting the fifth carbon to a carbonyl group areknown in the art and such embodiments are within the purview of theinvention.

Further embodiments of the invention also provide methods for producingamine containing OF-Δxyl analogue (FIG. 2B). In certain suchembodiments, an amine containing OF-Δxyl analogue is produced from analdehyde containing OF-Δxyl analogue. For example, an aldehydecontaining OF-Δxyl analogue is subject to a reductive amination with aprimary or secondary amine to produce an amine containing OF-Δxylanalogue. This is a two-step reaction, with a first step involving animine formation with an alcohol, such as MeOH, followed by reduction,for example, with NaBH₄ (FIG. 3B). Amines suitable for reductiveamination include undecylamine, dodecylamine and DL-dihydrosphingosine.

Additional reactions suitable for converting aldehyde containing OF-Δxylanalogue to amine containing OF-Δxyl analogue are well known to a personskilled in the art and such embodiments are within the purview of theinvention.

Further embodiments of the invention provide modifying amine containingOF-Δxyl analogues, particularly, at R₄ and R₅ positions of Formula III.For example, R₄ and R₅ in Formula III can be H, OH, a substituted orunsubstituted alkane, substituted or unsubstituted alkene, substitutedor unsubstituted alkyne, substituted or unsubstituted aryl, substitutedor unsubstituted heterocycle, substituted or unsubstituted heteroaryl,substituted or unsubstituted heterocycle, substituted or unsubstitutedether, substituted or unsubstituted thioether, substituted orunsubstituted ketone or a halogen. A skilled artisan can designreactions for converting amine containing OF-Δxyl analogues tocorresponding analogues containing such substitutions and suchembodiments are within the purview of the invention.

Further embodiments of the invention also provide methods for producinghydrazone containing OF-Δxyl analogue of Formula V (FIG. 2D). An exampleof such reaction is provided in FIG. 3C. In this reaction, an aldehydecontaining OF-Δxyl analogue is incubated with an alcohol, for example,methanol, at an appropriate temperature, for example, about 20 to 35°C., preferably, about 25° C., for about 40 to 60 hours, preferably,about 50 hours, even more preferably, about 48 hours. This reactionproduces an intermediate OF-Δxyl analogue, which is reduced with anappropriate reducing agent, for example, with NaBH₄ (FIG. 3C). Triazolemoiety illustrated in FIG. 3C can be substituted or unsubstituted on oneor more positions, for example, by OH, a substituted or unsubstitutedalkane, substituted or unsubstituted alkene, substituted orunsubstituted alkyne, substituted or unsubstituted aryl, substituted orunsubstituted heterocycle, substituted or unsubstituted heteroaryl,substituted or unsubstituted heterocycle, substituted or unsubstitutedether, substituted or unsubstituted thioether, substituted orunsubstituted ketone or a halogen.

Even further embodiments of the invention also provide methods forproducing hydrazone containing OF-Δxyl analogue of Formula V (FIG. 2D).In certain embodiments, a hydrazone containing OF-Δxyl analogue isproduced from an aldehyde containing OF-Δxyl analogue. An example ofsuch reaction is provided in FIG. 3D. In this reaction, an aldehydecontaining OF-Δxyl analogue is incubated with an alcohol, for example,methanol, at an appropriate temperature, for example, about 20 to 35°C., preferably, about 25° C., for about 10 to 20 hours, preferably,about 15 hours, even more preferably, about 12 hours. This reactionproduces an intermediate OF-Δxyl analogue, which is reduced with anappropriate reducing agent, for example, with NaBH₄ (FIG. 3D). R₄ or R₅in FIG. 3D can be independently H, OH, a substituted or unsubstitutedalkane, substituted or unsubstituted alkene, substituted orunsubstituted alkyne, substituted or unsubstituted aryl, substituted orunsubstituted heterocycle, substituted or unsubstituted heteroaryl,substituted or unsubstituted heterocycle, substituted or unsubstitutedether, substituted or unsubstituted thioether, substituted orunsubstituted ketone or a halogen.

In certain such embodiments, an aldehyde containing OF-Δxyl analogue istreated with certain hydrazines, for example, phenyl hydrazine,2-nitrophenyl hydrazine, 4-nitrophenyl hydrazine or 2,4-dinitrophenylhydrazine. In certain such embodiments, an appropriate hydrazine isdissolved in an aqueous organic reaction mixture in the presence of asuitable Bronsted acid or base or a suitable Lewis acid or base toproduce hydrazine containing OF-Δxyl analogues. Aqueous organic reactionmixtures suitable in such embodiments include mixtures ofacetonitrile/water, water/methanol and water/isopropanol. A skilledartisan can determine an appropriate ratio of water and the organicsolvent. Accordingly, under certain embodiments R₆ group of Formula Vcan be phenyl, 2-nitrophenyl, 4-nitrophenyl or 2,4-dinitrophenyl.

When present, the nitro-groups on the hydrazone containing OF-Δxylanalogues, for example, analogs made from 2-nitrophenyl hydrazine,4-nitrophenyl hydrazine or 2,4-dinitrophenyl hydrazine, can be furtherreduced to amines, which in turn could be further modified. For example,the resulting aromatic amines can be subjected to reductive aminationwith a variety of aliphatic or aromatic aldehydes, such as acetaldehyde,linear chain aldehydes, branched chain alkyl aldehydes and benzaldehyde.Additional modifications include conjugation to OH, a substituted orunsubstituted alkane, substituted or unsubstituted alkene, substitutedor unsubstituted alkyne, substituted or unsubstituted aryl, substitutedor unsubstituted heterocycle, substituted or unsubstituted heteroaryl,substituted or unsubstituted heterocycle, substituted or unsubstitutedether, substituted or unsubstituted thioether, substituted orunsubstituted ketone or a halogen. A skilled artisan can designreactions for converting amines from reduced hydrazone containingOF-Δxyl analogues to corresponding analogues containing suchsubstitutions and such embodiments are within the purview of theinvention.

Salts of Occidiofungin Analogues of the Invention

In some embodiments the subject invention provides salts of the OF-Δxylanalogues described herein. The salts can be a salt with an inorganicacid, such as hydrochloric acid, hydrobromic acid, perchloric acid,nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid; anorganic acid, such as trifluoroacetic acid (TFA), formic acid, aceticacid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalicacid, malonic acid, succinic acid, maleic acid, and fumaric acid; or asalt with a base, such as sodium hydroxide, ammonium hydroxide,potassium hydroxide, and organic bases such as mono-, di-, trialkyl andaryl amines, and substituted ethanolamines.

Further salts include: (1) acid addition salts, formed with inorganicacids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitricacid, phosphoric acid, and the like; or formed with organic acids suchas acetic acid, propionic acid, hexanoic acid, cyclopentanepropionicacid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinicacid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-di sulfonicacid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like; or (2)salts formed when an acidic proton present in the parent occidiofunginanalogue is replaced by a metal ion, e.g., an alkali metal ion, analkaline earth ion, or an aluminum ion; or coordinates with an organicbase such as ethanolamine, diethanolamine, triethanolamine,N-methylglucamine and the like. Salts further include, by way of exampleonly, sodium, potassium, calcium, magnesium, ammonium,tetraalkylammonium, and the like; and when the occidiofungin analoguescontains a basic functionality, salts of non-toxic organic or inorganicacids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate,maleate, oxalate and the like.

Certain embodiments provide amorphous forms of salts of the OF-Δxylanalogues disclosed herein. Such amorphous forms are advantageous fororal, pulmonary, buccal, suppository delivery.

Routes of Administration and Dosage Forms

In certain embodiments, the OF-Δxyl analogues can be administeredintramuscularly, subcutaneously, intrathecally, intravenously orintraperitoneally by infusion or injection. Solutions of theoccidiofungin analogues can be prepared in water, optionally mixed witha nontoxic surfactant. Under ordinary conditions of storage and use,these preparations can contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the occidiofungin analogues that are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. Preferably, theultimate dosage form should be sterile, fluid, and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained by, for example, the formation of liposomes, by themaintenance of the required particle size in the case of dispersions, orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it is preferable toinclude isotonic agents, for example, sugars, buffers, or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the OF-Δxylanalogues in the required amount in the appropriate solvent as describedherein with various of the other ingredients enumerated herein, asrequired, preferably followed by filter sterilization. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze dryingtechniques, which yield a powder of the active ingredient plus anyadditional desired ingredient present in the previously sterile-filteredsolutions.

The compositions of the subject invention may also be administeredorally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the subject'sdiet.

For oral therapeutic administration, the OF-Δxyl analogues can becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. Such compositions and preparations shouldcontain at least 0.1% of a occidiofungin analogue of the presentinvention. The percentage of the occidiofungin analogues of theinvention present in such compositions and preparations may, of course,be varied and may conveniently be between about 2% to about 60% of theweight of a given unit dosage form. The amount of the OF-Δxyl analoguesin such therapeutically useful compositions is such that an effectivedosage level can be obtained.

The tablets, troches, pills, capsules, and the like may also contain oneor more of the following: binders such as gum tragacanth, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acid,and the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, fructose, lactose, or aspartame, or a flavoringagent such as peppermint, oil of wintergreen, or cherry flavoring may beadded.

When the unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol.

Various other materials may be present as coatings or for otherwisemodifying the physical form of the solid unit dosage form. For instance,tablets, pills, or capsules may be coated with gelatin, wax, shellac, orsugar, and the like. A syrup or elixir may contain the an occidiofunginanalogue, sucrose or fructose as a sweetening agent, methyl andpropylparabens as preservatives, a dye, and flavoring such as cherry ororange flavor.

Of course, any material used in preparing any unit dosage form should bepharmaceutically acceptable and substantially non-toxic in the amountsemployed.

In addition, the OF-Δxyl analogues may be incorporated intosustained-release preparations and devices. For example, theoccidiofungin analogues may be incorporated into time release capsules,time release tablets, time release pills, and time release occidiofunginanalogues or nanoparticles.

Pharmaceutical compositions for topical administration of the OF-Δxylanalogues to the epidermis (mucosal or cutaneous surfaces) can beformulated as ointments, creams, lotions, gels, or as a transdermalpatch. Such transdermal patches can contain penetration enhancers suchas linalool, carvacrol, thymol, citral, menthol, t-anethole, and thelike. Ointments and creams can, for example, include an aqueous or oilybase with the addition of suitable thickening agents, gelling agents,colorants, and the like. Lotions and creams can include an aqueous oroily base and typically also contain one or more emulsifying agents,stabilizing agents, dispersing agents, suspending agents, thickeningagents, coloring agents, and the like. Gels preferably include anaqueous carrier base and include a gelling agent such as cross-linkedpolyacrylic acid polymer, a derivatized polysaccharide (e.g.,carboxymethyl cellulose), and the like.

Pharmaceutical compositions suitable for topical administration in themouth (e.g., buccal or sublingual administration) include lozengescomprising the composition in a flavored base, such as sucrose, acacia,or tragacanth; pastilles comprising the composition in an inert basesuch as gelatin and glycerin or sucrose and acacia; and mouthwashescomprising the active ingredient in a suitable liquid carrier. Thepharmaceutical compositions for topical administration in the mouth caninclude penetration enhancing agents, if desired.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina, and the like. Other solidcarriers include nontoxic polymeric nanoparticles or microparticles.Useful liquid carriers include water, alcohols, or glycols, orwater/alcohol/glycol blends, in which the occidiofungin analogues can bedissolved or dispersed at effective levels, optionally with the aid ofnon-toxic surfactants. Adjuvants such as fragrances and additionalantimicrobial agents can be added to optimize the properties for a givenuse. The resultant liquid compositions can be applied from absorbentpads, used to impregnate bandages and other dressings, or sprayed ontothe affected area using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses, or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the OF-Δxyl analogues to the skin are known in the art; forexample, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat.No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman(U.S. Pat. No. 4,820,508), all of which are hereby incorporated byreference.

The concentration of the OF-Δxyl analogues of the invention in suchformulations can vary widely depending on the nature of formulation andintended route of administration. For example, the concentration of theoccidiofungin analogues in a liquid composition, such as a lotion, canpreferably be from about 0.1-25% by weight, or, more preferably, fromabout 0.5-10% by weight. The concentration in a semi-solid or solidcomposition such as a gel or a powder can preferably be about 0.1-5% byweight, or, more preferably, about 0.5-2.5% by weight.

Pharmaceutical compositions for spinal administration or injection intoamniotic fluid can be provided in unit dose form in ampoules, pre-filledsyringes, small volume infusion, or in multi-dose containers, and caninclude an added preservative. The compositions for parenteraladministration can be suspensions, solutions, or emulsions, and cancontain excipients such as suspending agents, stabilizing agents, anddispersing agents.

A pharmaceutical composition suitable for rectal administrationcomprises OF-Δxyl analogues of the present invention in combination witha solid or semisolid (e.g., cream or paste) carrier or vehicle. Forexample, such rectal compositions can be provided as unit dosesuppositories. Suitable carriers or vehicles include cocoa butter andother materials commonly used in the art.

According to one embodiment, pharmaceutical compositions of the presentinvention suitable for vaginal administration are provided as pessaries,tampons, creams, gels, pastes, foams, or sprays containing anoccidiofungin analogue of the invention in combination with carriers asare known in the art. Alternatively, compositions suitable for vaginaladministration can be delivered in a liquid or solid dosage form.

Pharmaceutical compositions suitable for intra-nasal administration arealso encompassed by the present invention. Such intra-nasal compositionscomprise an occidiofungin analogue of the invention in a vehicle andsuitable administration device to deliver a liquid spray, dispersiblepowder, or drops. Drops may be formulated with an aqueous or non-aqueousbase also comprising one or more dispersing agents, solubilizing agents,or suspending agents. Liquid sprays are conveniently delivered from apressurized pack, an insufflator, a nebulizer, or other convenient meansof delivering an aerosol comprising an occidiofungin analogue.Pressurized packs comprise a suitable propellant such asdichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide, or other suitable gas as iswell known in the art. Aerosol dosages can be controlled by providing avalve to deliver a metered amount of an occidiofungin analogue.

The OF-Δxyl analogues may be combined with an inert powdered carrier andinhaled by the subject or insufflated.

Pharmaceutical compositions for administration by inhalation orinsufflation can be provided in the form of a dry powder composition,for example, a powder mix of an occidiofungin analogue and a suitablepowder base such as lactose or starch. Such powder composition can beprovided in unit dosage form, for example, in capsules, cartridges,gelatin packs, or blister packs, from which the powder can beadministered with the aid of an inhalator or insufflator.

The exact amount (effective dose) of the OF-Δxyl analogues varies fromsubject to subject, depending on, for example, the species, age, weight,and general or clinical condition of the subject, the severity ormechanism of any infection being treated, the particular agent orvehicle used, the method and scheduling of administration, and the like.A therapeutically effective dose can be determined empirically, byconventional procedures known to those of skill in the art. See, e.g.,The Pharmacological Basis of Therapeutics, Goodman and Gilman, eds.,Macmillan Publishing Co., New York. For example, an effective dose canbe estimated initially either via in vivo assays or in suitable animalmodels. The animal model may also be used to determine the appropriateconcentration ranges and routes of administration. Such information canthen be used to determine useful doses and routes for administration inhumans. Methods for the extrapolation of effective dosages in mice andother animals to humans are known to the art; for example, see U.S. Pat.No. 4,938,949, which is hereby incorporated by reference. A therapeuticdose can also be selected by analogy to dosages for comparabletherapeutic agents.

The particular mode of administration and the dosage regimen can beselected by the attending clinician, taking into account the particularsof the case (e.g., the subject, the disease, the disease state involved,and whether the treatment is prophylactic). Treatment may involve dailyor multi-daily doses of compound(s) over a period of a few days tomonths, or even years.

In general, however, a suitable dose can be in the range of from about0.001 to about 100 mg/kg of body weight per day, preferably from about0.01 to about 100 mg/kg of body weight per day, more preferably, fromabout 0.1 to about 50 mg/kg of body weight per day, or even morepreferred, in a range of from about 1 to about 10 mg/kg of body weightper day. For example, a suitable dose may be about 1 mg/kg, 10 mg/kg, or50 mg/kg of body weight per day.

The OF-Δxyl analogues can be conveniently administered in unit dosageform, containing for example, about 0.05 to about 10000 mg, about 0.5 toabout 10000 mg, about 5 to about 1000 mg, or about 50 to about 500 mg ofactive ingredient per unit dosage form.

The OF-Δxyl analogues can be administered to achieve peak plasmaconcentrations of, for example, from about 0.25 to about 200 μM, about0.5 to about 75 μM, about 1 to about 50 μM, about 2 to about 30 μM, orabout 5 to about 25 μM. Exemplary desirable plasma concentrationsinclude at least 0.25, 0.5, 1, 5, 10, 25, 50, 75, 100 or 200 μM. Forexample, plasma levels may be from about 1 to about 100 micromolar orfrom about 10 to about 25 micromolar. This may be achieved, for example,by the intravenous injection of a 0.05 to 5% solution of an OF-Δxylanalogue, optionally in saline, or orally administered as a boluscontaining about 1 to about 100 mg of the OF-Δxyl analogue. Desirableblood levels may be maintained by continuous or intermittent infusion.

The OF-Δxyl analogues can be included in the compositions within atherapeutically useful and effective concentration range, as determinedby routine methods that are well known in the medical and pharmaceuticalarts. For example, a typical composition can include one or more of theOF-Δxyl analogues at a concentration in the range of at least about 1mg/ml, preferably at least about 4 mg/ml, more preferably at least 5mg/ml and most preferably at least 6 mg/ml.

The OF-Δxyl analogues may conveniently be presented in a single dose oras divided doses administered at appropriate intervals, for example, asone dose per day or as two, three, four or more sub-doses per day. Thesub-dose itself may be further divided, e.g., into a number of discreteloosely spaced administrations; such as multiple inhalations from aninsufflator.

Optionally, the pharmaceutical compositions of the present invention caninclude one or more other therapeutic agents, e.g., as a combinationtherapy. The additional therapeutic agent(s) will be included in thecompositions within a therapeutically useful and effective concentrationrange, as determined by routine methods that are well known in themedical and pharmaceutical arts. The concentration of any particularadditional therapeutic agent may be in the same range as is typical foruse of that agent as a monotherapy, or the concentration may be lowerthan a typical monotherapy concentration if there is a synergy whencombined with an occidiofungin analogue of the present invention.

Methods of Treatment

As discussed above, the OF-Δxyl analogues disclosed herein exhibitanti-fungal activity, particularly, against Candida spp. Accordingly,certain embodiments of the invention provide methods of administering anOF-Δxyl analogue to a subject in need thereof to treat or prevent aninfection, particularly, a fungal infection, more particularly, a yeastinfection, and even more particularly, a Candida infection. In specificembodiments, the invention provides methods of treating or preventing aninfection caused by a fungus selected from Trichophyton mentagrophytes,Trichophyton rubrum, Rhizopus microspores, Mucor circinelloides, Mucorfragilis, Fusarium solani, Fusarium oxysporum, Aspergillus flavus,Aspergillus fumigatus, Candida albicans, Candida glabrata, Candidakrusei, Candida krusei, Candida parapsilosis, Candida tropicalis orCryptococcus neoformans. Particularly, methods are provided for treatingor preventing an infection with any strain listed in Table 8.

Methods for treating or preventing an infection can be performed in anysubject, such as a mammal, including humans. Such methods compriseadministering to a subject in need of such prevention or treatment of aninfection an effective amount of an OF-Δxyl analogue the subjectinvention. An OF-Δxyl analogue can be administered in the form of apharmaceutical composition of an OF-Δxyl analogue.

Preferably, an occidiofungin analogue is administered parenterally orenterally. Even more preferably, an occidiofungin analogue isadministered intravaginally, intraperitoneally, subcutaneously orintravenously. The dosage of the effective amount of an occidiofunginanalogue can vary depending upon the age and condition of each subjectto be treated. However, suitable unit dosages typically range from about0.01 to about 100 mg. For example, a unit dose can be in the range ofabout 0.2 mg to about 50 mg. Such a unit dose can be administered morethan once a day, e.g., two or three times a day.

Materials and Methods

Occidiofungin was purified as previously described by Lu et al. Purifiedrabbit skeletal muscle filamentous actin (AKF99) was purchased fromCytoskeleton Inc. (Denver, Colo.). Sodium periodate (311448-5G), sodiumborohydride (452882), undecylamine (94200-10ML), dodecylamine(325163-5ML), and DL-dihydrosphingosine (D6783-10MG) were purchased fromSigma (St. Louis, Mo.).

Synthesis of Occidiofungin Analogs

Occidiofungin was dissolved in 50% acetonitrile (ACN) with 0.1%trifluoroacetic acid (TFA) at a concentration of 1 mg/mL. Sodiumperiodate was dissolved in ddH₂O at a concentration of 1 mg/mL. Equalvolumes of the two solutions were mixed thoroughly in a 10 mL centrifugetube and was incubated at 70° C. for 30 min. One milliliter of thereaction mixture was loaded on a BioRad Duoflow chromatography systemwith an analytical C18 column (Agilent® ZORBAX, (Agilent Technologies,Santa Clara, Calif. ODS, C18, 5 μm, 4.6×250 mm). The reaction mixturewas separated with an isocratic flow of 20% MeOH in water and theresulting aldehyde analog of occidiofungin eluted at approximately 9minutes. The desired product was freeze-dried and weighed on ananalytical balance (Adventurer™ Pro AV114C, Ohaus Corporation, USA).

A two-step procedure of reductive amination was used to introduce aprimary or secondary amine onto the aldehyde analog. The first step ofreaction involves the formation of an intermediate carbinol amine, whichis then dehydrated and protonated to form an iminium ion. Subsequentreduction of this iminium ion with sodium borohydride produces analkylated amine product. The aldehyde analog of occidiofungin wasdissolved in DMSO at a concentration of 10 mg/mL. Amines used wereundecylamine, dodecylamine, and DL-dihydrosphingosine. Aldehyde analog(10 μL of DMSO stock solution; 100 μg) was mixed with 10-fold excess ofan amino lipid (molar ratio) in 400 μL methanol. The sample was thenincubated at room temperature for at least 16 hours. Solid sodiumborohydride (6 mg) was weighted on an analytical balance (Adventurer™Pro AV114C, Ohaus Corporation, USA) and was directly transferred intothe reaction mixture, mixed well, and was incubated at room temperaturefor 5 minutes. The reaction mixture was diluted to 1 mL with 50% ACN0.1% TFA and was separated by HPLC using a 30-minute gradient from 90%to 20% water/ACN on the analytical C18 column. Both HPLC solventscontained 0.1% TFA. Products that eluted between 50-30% water werecollected and analyzed by electrospray ionization mass spectrometry(ESI-MS) on a ThermoFisher DecaXP ion trap mass spectrometer. The yieldof each sample was quantified by comparing the peak area of each analogto the peak area of a 100 μg standard of occidiofungin.

NMR Spectroscopy

NMR analysis of aldehyde occidiofungin analog was performed on a 5 mMsample dissolved in dimethyl sulfoxide (DMSO)-d6. The NMR data werecollected on an Avance III HD-600 with a TCI Cryoprobe and an Avance IIIHD-850 with a TCI Cryoprobe. The ¹H resonances were assigned accordingto standard methods using COSY, TOCSY, NOESY, and ¹³C-HSQC experiments.NMR experiments were collected at 25° C. ¹H chemical shifts werereferenced to DMSO peak at 2.5 ppm. The TOCSY experiment was acquiredwith a 60 ms mixing time using the Bruker DIPSI-2 spinlock sequence. TheNOESY experiment was acquired with a 400 ms mixing time. Phase sensitiveindirect detection for NOESY, TOCSY, and COSY experiments was achievedusing the standard Bruker pulse sequences. Peaks were assigned usingNMRView.

Minimum Inhibitory Concentration Assays

The minimal inhibitory concentration (MIC) is the lowest concentrationof compound that inhibits the visible growth of the yeast after 24 hoursof incubation. MIC assays were performed in duplicate as previouslydescribed following a modified version of the CLSI M27-A3 methods.

Actin Co-Sedimentation Assay

Actin binding experiments were performed as previously described fornative occidiofungin by Ravichandran et al. An Agilent 1200 front endchromatography system and a TSQ Quantum™ Access Triple Quadrupole MassSpectrometer was used to analyze phalloidin, native occidiofungin,dodecylamine analog, and the DL-dihydrosphingosine analog binding toF-actin. Following a 10 μL injection, samples containing dodecylamineanalog of occidiofungin were separated using a 15-minute water/ACN(containing 0.2% formic acid) gradient starting from 95% to 40% water ona C18 column (SinoChrom ODS-BP 5 μm, 2.1 mm×50 mm). Samples containingDL-dihydrosphingosine analog were separated on the same column through amodified water/ACN gradient starting from 95% to 56% water over 5minutes, from 56% to 52% water over 2 minutes, followed by a lineargradient from 52% to 48% water over 1 minute. The mass spectrometer wasoperated in positive mode and operated using a protocol optimized foreach compound. Briefly, the center mass for dodecylamine analog was1037.66 Da and the scan width was 0.3 Da. The center mass fordihydrosphingosine analog was 1153.75 Da and the scan width was 1.0 Da.Area of each compound was measured through manual integration usingXcalibur™ Software (Thermo Fisher Scientific). The standard curves weregenerated for each compound following the extraction protocol describedabove. Native occidiofungin served as the internal standard for allanalogs. The R² values for each standard curve exceeded 0.98.

Actin Polymerization and Depolymerization Assay

Actin polymerization and depolymerization assays were performed usingthe Actin polymerization biochem kit (#BK003; Cytoskeleton, Inc. Denver,Colo.) following the manufacturer's instructions with somemodifications. Briefly, for the polymerization assay, G-actin stocksolution was made at 0.2 mg/mL instead of 0.4 mg/mL. For thedepolymerization assay, F-actin stock solution was diluted 9-foldinstead of 5-fold. Test compounds include phalloidin, dihydrosphingosineanalog, and native occidiofungin, at a final concentration of 5 μM. Theexperiments were done in duplicate.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example 1—Synthesis of Semisynthetic Analogues of Occidiofungin

Occidiofungin was produced and purified as described by Lu et al. andEmrick et al. Chemicals were purchased from Sigma unless otherwiseindicated. Wild-type MS14 strain and xylose transferase mutant strainused were obtained from glycerol stocks and cultured on Yeast PeptoneDextrose (YPD) agar plate or broth at 35° C. Occidiofungin was producedfrom these cultures and used to produce OF-Δxyl analogues as describedherein.

Cleavage of diols on carbons 5 and 6 of the novel amino acid yields analdehyde containing OF-Δxyl analogue. Sodium periodate breaks apartvicinal diols to form an aldehyde containing analogue of OF-Δxyl (FIGS.3A-3B). Occidiofungin was dissolved in 50% ACN with 0.1% TFA at aconcentration of 1 mg/mL. Sodium periodate was dissolved in ddH₂O at aconcentration of 1 mg/mL. Equal volume of the two solutions were mixedin a centrifuge tube and incubated at various temperatures from 30-70°C. for 30 min. The reaction mixture was loaded on a BioRad Duoflow™chromatography system with an analytical column (Agilent® ZORBAX,Agilent Technologies, Santa Clara, Calif. ODS, C18, 5 μm, 4.6×250 mm).The reaction mixture was chromatographed with an isocratic flow of 20%MeOH in ddH₂O. The aldehyde containing analogue of OF-Δxyl elutes atapproximately 9 minutes (FIG. 4). The desired product is dried andweighed on analytical balance (Adventurer™ Pro AV114C, OhausCorporation, USA). The efficiency of the reaction and recovery of thedesired aldehyde-occidiofungin product was greater than 90%.

Reductive amination restores the aliphatic structural element ofoccidiofungin through a condensation reaction with the free amine. Atwo-steps procedure of reductive amination of aldehyde containingOF-Δxyl analogue (FIG. 2A) was used to introduce a primary or secondaryamine at aldehyde. The first step of reaction involves the formation ofan intermediate carbinol amine, which then undergoes dehydratation andprotonation to form an iminium ion. Subsequent reduction of this iminiumion with sodium borohydride produces an alkylated secondary or tertiaryamine product depending on the nature of the starting material.

Chemicals used produce certain OF-Δxyl analogues include undecylamine,dodecylamine, and DL-dihydrosphingosine. For example, aldehydecontaining OF-Δxyl analogue is dissolved in DMSO at a concentration of10 mg/mL and mixed with 10-fold excess of undecylamine (molar ratio) inpure methanol and was incubated at room temperature overnight. Solidsodium borohydride (6 mg) was added to the overnight reaction andincubated at room temperature for 5 minutes. The resulting product inthe reaction mixtures was chromatographically purified using a linearwater/ACN gradient starting from 90% to 20% water over 30-minute period.Both solvents used in the mobile phase contained 0.1% trifluoroaceticacid (TFA). The products eluted between 50-30% water with 0.1% TFA (FIG.5). The reaction efficiencies were determined by comparing the peak areaof each sample to the peak area of a 100 μg standard of occidiofungin(Table 1). The efficiency of the reactions and isolation of the desiredproducts were greater than 50% for all reactions.

The molecular weight of the purified product was determined byelectrospray ionization mass spectrometry (ESI-MS) on a ThermoFisherDecaXP ion trap mass spectrometer (Table 1). Briefly, around 1 μg ofdried sample was resuspended in 100 μl of 50% ACN/water (vol/vol) andanalyzed by direct infusion in positive mode. MS data were recorded in a0.5 min time frame (FIG. 6). Occidiofungin wild-type was used as acomparison and mass spectral control. Nuclear magnetic resonance studieswere done to verify the structure of the OF-Δxyl analogue.

TABLE 1 Summary of reactions. The starting material, type of reaction,and yield of reaction are shown. The products are confirmed by ESI-MS,all measured mass are the same with expected. Yield of oxidationreaction was determined based on dry weight of both starting materialand product, while the yields of reductive amination reactions werebased on the peak volume of both starting material and product on HPLC.Molecular Weight Starting Type Yield (Da) material of reaction Amine (%)Expected Measured wild-type oxidation N.A. 90 868 868 aldehyde reductiveundecylamine 70 1024 1024 variant amination aldehyde reductivedodecylamine 84 1038 1038 variant amination aldehyde reductiveDL-dihydro- 51 1154 1154 variant amination sphingosine

The removal of the aliphatic chain resulted in a highly polar compound(FIGS. 1-2), which was isolated by HPLC under isocratic conditions.Oxidative cleavage of the vicinal diols proceeded efficiently, andoccidiofungin was completely converted to the aldehyde product (FIG. 2and FIG. 3A). This was confirmed by mass spectrometry of the reactionmixture (FIG. 4B). The isolated aldehyde has a mass of 868 Daltons (Da).Nuclear magnetic resonance (NMR) analyses of the isolated productfurther confirmed the isolation of the cyclic peptide with the aldehydeon carbon 5 (C5) of the NAA2 residue. The individual amino acid spinsystems in the 2D TOCSY spectra are shown in (FIG. 8A) and the chemicalshifts for the aldehyde product are provided in Table 2. Thethrough-space proton interaction between the aldehyde proton and theamide proton of the novel amino acid was observed in the 2D NOESYspectra (FIG. 8B), supporting the proton chemical shift assignments ofthe aldehyde product. The aldehyde occidiofungin product wassubsequently used in reductive amination reactions affording thesynthesis of new analogs of occidiofungin.

Reductive amination reactions using three commercially available longchain alkyl amines and the aldehyde occidiofungin products wereperformed (FIG. 3B). The oxidative cleavage of the diol in the NAA2resulted in a loss of thirteen carbons in the aliphatic chain. Since thexylose itself is not required for the antifungal activity, undecylamineand dodecylamine were used to restore the length of the aliphatic chain.A secondary amine is incorporated at the carbon six (C6) position and analiphatic chain of eleven and twelve carbons were introduced for theundecylamine and dodecylamine products, respectively (FIG. 3E). Thealkyl amine dihydrosphingosine was chosen because a hydroxyl group wouldbe reintroduced into the side chain near the normally occurringpositions found within the native product (FIG. 3E). The unoptimizedreactions were very efficient. The yield of the semisyntheticundecylamine, dodecylamine, and dihydrosphingosine analogs were 70, 80,and 50%, respectively (Table 2, FIGS. 5-6). The final undecylamine,dodecylamine, and dihydrosphingosine occidiofungin products had theexpected masses of 1023.7, 1037.7, and 1153.9 Da, respectively (Table 2,FIG. 5-6). Additional analogs can be rapidly synthesized by thisapproach and the structural diversity of analogs made can be expanded bythe synthesis of novel amino lipid compounds that are not readilyavailable.

TABLE 2 NMR chemical shifts for aldehyde analog of occidiofungin. Protonchemical shift values were from a TOCSY and NOESY experiments. AminoAcid H^(N) H^(α) H^(β) Other protons BHN1 8.11 4.57 4.09 β-OH: 5.68,γ-NH2: 7.20, 6.70 NAA2 7.76 C2: CH2- 7.58 2.08&1.92, C3: CH- 4.39, C4:CH2-2.96, C5: CH-8.71 Ser3 8.12 4.27 3.56 β-OH: 4.99 BHY4 8.02 4.45 4.04β-OH: 5.71, OH-9.28, C2&C6: CH-7.16, C3&C5: CH-6.70 DABA5 7.61 4.23 2.02γ-H: 2.91, NH2: 7.71 γ-H: 2.87, NH2: 7.66 Gly6 8.07 3.82, 3.64 Asn7 8.164.59 2.58, 2.47 γ-NH2: 7.44, 6.97 Ser8 8.01 4.20 3.61 β-OH: 5.08

Example 2—Minimum Inhibitory Concentration (Mic) of the OF-ΔxylAnalogues

Minimum inhibitory concentration (MIC) susceptibility testing wasperformed following a modified version, as previously reported, of theCLSI M27-A3 methods for the susceptibility testing of yeasts. Incubationtemperature was 35° C. and the inoculum size was 0.5-2.5×10³colony-forming units (CFU)/mL for yeasts. MICs were performed todetermine the bioactivity of each new OF-Δxyl analogue (Table 3). Theminimal inhibitory concentration (MIC) is the lowest concentration ofcompound that inhibits the visible growth of the yeast after 24 hours ofincubation and the assays were performed in duplicate. The aldehydecontaining OF-Δxyl analogues with the addition of an undecylamine,dodecylamine, or DL-dihydrosphingosine did not demonstrate anyinhibitory activity against C. albicans at the concentration tested.However, the addition of DL-dihydrosphingosine to the aldehydecontaining OF-Δxyl analogue exhibited a low micromolar inhibitoryactivity against Saccharomyces cerevisiae and Candida glabrata.

The aldehyde, undecylamine, and dodecylamine analogs were inactive atconcentrations 64-fold higher than the inhibitory concentration ofnative occidiofungin against the Candida species tested and S.cerevisiae strain tested (Table 3). The undecylamine and dodecylamineanalogs have a similar aliphatic carbon length as native occidiofungin.Further, these analogs have similar polarity as native occidiofungin, aswas observed by the HPLC retention times (FIGS. 5-6). The majorstructural differences to native compound are the conversion of a(C5)-(C6) to (C5)-(N6) bond in the side chain of NAA2, the removal ofthe vicinal diol group, and the loss of a xylose sugar.Dihydrosphingosine analog was synthesized to test whether theintroduction of a hydroxyl group near (C6) position could restoreactivity. Dihydrosphingosine analog has one branched alcohol on (C7) anda hydroxyl on (C8) positions normally found within native occidiofungin.Further, the aliphatic chain of dihydrosphingosine analog is fivecarbons longer than the native compound. The dihydrosphingosine restoressome antifungal activity as observed by a low micromolar inhibitoryactivity against S. cerevisiae, C. albicans, and Candida glabrata; theMICs were 2, 16, and 8 μg/mL, respectively. The loss of activity, or thereduction of activity, of the semisynthetic analogs of occidiofungincould be attributed to a reduction in their ability to penetrate theplasma membrane to reach cellular target (binding to cell envelope orinability to cross plasma membrane) or a loss in affinity to actin (thecellular target of occidiofungin).

TABLE 3 MIC (μg/ml) of occidiofungin analogues against Candida glabrataATCC2001, Candida albicans ATCC 3147 and S. cerevisiae DGY6 haploidBY4741. Sacchaomyces cerevisiae Candida occidiofungin DGY6 haploidglabrata Candida albicans analogues BY4741 ATCC 2001 ATCC 3147 wild-type0.0625 0.5 0.5 aldehyde containing >4 — >8 OF-Δxyl analogueUndecylamine >4 — >8 containing OF-Δxyl analogue Dodecylamine >4 — >8containing OF-Δxyl analogue DL-dihydrosphingosine 2 8 >8 containingOF-Δxyl analogue

Example 3—Additional Examples OF-Δxyl Analogues

The aldehyde containing OF-Δxyl analogue can be used to create newanalogues of occidiofungin besides the amine containing OF-Δxylanalogues described above. Synthesis of triazoles can be readilyaccomplished by addition of various aryl azide in the presence ofCu(ACAC) as a catalyst. This allows for the introduction of various arylgroups and conversion of the aldehyde into a triazole.

Synthesis of hydrazones: The aldehyde occidiofungin (1) can be treatedwith a variety of phenyl hydrazines such as i) 2-nitrophenyl hydrazine,4-nitrophenyl hydrazine or 2,4-dinitrophenyl hydrazine (2,4 DNPH) toproduce novel hydrazones. The nitro groups on these hydrazones can bereadily reduced using a variety of reducing agents such as NaBH4/Pd,Pd/H2 and stannous chloride to produce the corresponding amines. Theresulting aromatic amines can be subjected to reductive amination with avariety of aliphatic or aromatic aldehydes, such as acetaldehyde, linearchain aldehydes, branched chain alkyl aldehydes and benzaldehyde.

Example 4—Efficacy of OF-Δxyl Analogues for Treating VVC

Six to eight-week-old BALB/c mice would be intravaginally infected withC. albicans and dosed once per day with OF-Δxyl analogues for two to sixdays, particularly, three days. The OF-Δxyl analogue treated groupswould be compared to a vehicle control group. Several groups of micewould be treated with various concentrations of OF-Δxyl analogues. Theoccidiofungin treated groups would be expected to reduce fungal load bymore than two logs compared to control groups and significantly betterthan naturally occurring occidiofungins. The mice would be examined foroutward signs of distress or irritation. No behavioral changes includingsluggishness, stretching, or reluctance to consume food is expected.Furthermore, no vaginal bleeding or swelling is expected followingOF-Δxyl analogue treatment.

The murine model of VVC has been reported by Yano et al. A variation ofthis method would be followed. Several groups of six mice would be usedto evaluate several concentrations of OF-Δxyl analogue and vehiclecontrol. Briefly, six to eight-week-old BALB/c mice would be treatedsubcutaneously with 200 ng per mouse of β-Estradiol 17-valerate threedays prior to inoculation with C. albicans (D-3). A subcutaneous dose ofestradiol would be administered every three days (D0, D3) until the endof the experiment to induce pseudo-estrus. Approximately a 20 μLintravaginal inoculation of a 2.5×10⁶ colony forming units (CFU)/mL ofC. albicans would define day zero (D0) of the VVC study. On the same dayof inoculation (D0), another subcutaneous injection of estradiol wouldbe made. OF-Δxyl analogues at several concentrations would be suspendedin 20 μL of warm 0.3% Noble agar before intravaginal inoculation. Drugtreatment would be done on day 2 (D2), day 3 (D3) and day 4 (D4) of thestudy. On day 5 (D5), the vaginal lumen would be lavaged with 100 μL ofsterile PBS with a 200 μL pipette tip. Serial dilutions and total colonyforming units per vaginal lavage would be determined by plating on YPDplates containing 50 μg/mL of chloramphenicol. The colony forming units(CFUs) obtained from each lavage would be counted on plates containing30-300 colonies for determining the CFU/mL estimates. Body weight, signsof vaginal irritation such as swelling or bleeding and clinical signs ofdiscomfort (stereotypical stretching behavior) would be monitored.Statistical analyses (T-test) would be done to compare the control groupto treated groups and to compare differences between treated groups.

Example 5—Methods of Treating Fungal Infections

Intraperitoneal (ip) and subcutaneous routes of administration:Toxicological analysis of occidiofungin was carried out using asuspension of occidiofungin in solvents such as phosphate bufferedsaline (PBS), PBS containing 0.5% methylcellulose and 0.1% Tween 20.Occidiofungin was not completely soluble in these solvents and suspendedparticles were observed in the solution. Sonication mitigated the amountof suspended particles but not entirely. Occidiofungin was administeredto female C57BI/6 C3H F1 (B6C3F1) mice at age of 6 to 8 weeks viamultiple routes and different doses to estimate toxicity.

Occidiofungin was administered by intraperitoneal injection in a singledose at 1, 5, 10, or 20 mg/kg in PBS of body weight. The compound wasalso administered in 1 dose at 10 mg/kg in sterilized 0.5%methylcellulose (constituted with 0.1% Tween-80 in PBS to promotesolubility of test agent) by ip or subcutaneous injection. The excipientcontrol in each experiment matched the vehicle. Control groups receivedsterile PBS or 0.5% methylcellulose with 0.1% Tween-80 in PBS. Bodyweight and clinical signs (movement, posture, skin lesions, appearanceof fur indicating normal grooming, and behaviors) were recorded dailyfor 5 days. Necropsies were performed on study day 1 or study day 5following the administration of occidiofungin. Blood and tissue sampleswere collected from animals dosed at 10 mg/kg with occidiofungin in 0.5%methylcellulose. One group of samples was collected on study day 1following a single dose by ip injection. Another group was collectedafter 5-day observation with the same single dose through subcutaneousinjection. Mice were anesthetized with isofluorane. Blood was then takenfrom the retroorbital plexus for serum biochemistry assays (alkalinephosphatase, alanine aminotransferase [ALT], aspartateaminotransferase[AST], gamma-glutamyl transferase, creatine kinase,blood urea nitrogen, creatinine) and hematology (white blood cell countand white blood cell differentiation). Body weight was measuredimmediately after mice were fully anesthetized and organs (spleen,thymus, brain, kidney, lung, and liver) were weighed and fixed in 10%neutral buffered formalin. Histological examination was performed on aportion of each organ by using routine paraffin-embedding technique andstaining with hematoxylin and eosin (H & E).

Occidiofungin was also evaluated in a 5-day toxicity study by using 5mice per group. Occidiofungin was dissolved in sterile PBS at dose of 2mg/kg and administrated by ip injection daily for 5 consecutive days.The control group received sterile PBS. Body weight and clinical signswere recorded daily. Necropsies were performed on day 5 following thefirst administration of occidiofungin. Clinical chemistry, hematology(the same parameters as in the single-dose study), and histology (thesame organs as in the single-dose study, except thymus) were evaluated.Histological sections were reviewed in the same manner as describedabove. Body weight, body weight change, organ weight, serum chemistry,and hematology data were analyzed by T test or 2-way analysis ofvariance (ANOVA) followed by Bonferroni posttest using Prism GraphPadsoftware (San Diego, Calif.). All the analyses were 2-sided, with P<0.05considered statistically significant.

Results of intraperitoneal (ip) and subcutaneous routes ofadministration are as follows. The body weight differences ranged from2% to 12% on the first day after treatment at various doses ofoccidiofungin in PBS (containing 0.5% methylcellulose or 0.2% Tween 20).At the end of the studies, it was clear that higher dose induced morebody weight loss, and this change was dose responsive. However, thiseffect tended to diminish after the dosing ended, with a consequentregain of body weight during the following days. Daily dosing at 2 mg/kgfor 5 days had a similar effect on day 5 as a single 5 mg/kg ip dose.Further, a decrease in absolute total white blood cell count in mice 1day after ip administration of occidiofungin at 10 mg/kg was observed.There were no statistical differences in hematological or serumchemistry values 5 days after subcutaneous administration ofoccidiofungin at 10 mg/kg (Table 4).

TABLE 4 Hematology and serum chemistry in single dose experiments usingoccidiofungin in PBS. Single Dose of Occidiofungin 0 mg/kg 10 mg/kg A:Hematology WBC (1000/μl) 3.5 ± 0.3  1.9 ± 0.3 * NEU (%) 16.3 ± 2.8  11.6± 1.4  LYM (%) 77.5 ± 3.7  80.4 ± 2.2  MONO (%) 3.3 ± 0.7 6.8 ± 1.7Serum Biochemistry ALP (U/l) 127.0 ± 2.0  67.0 ± 7.7 * ALT (U/l) 59.0 ±5.6  58.0 ± 2.6  AST (U/l) 110.0 ± 13.3  160.0 ± 26.2  GGT (U/l) 20.0 ±2.7  20.0 ± 1.6  CK (U/l) 146.3 ± 36.4  203.0 ± 31.6  BUN (mg/dl) 23.0 ±1.2  21.0 ± 1.0  CREAT (mg/dl) 0.1 ± 0.1 0.1 ± 0.1 Effect ofoccidiofungin on hematology and serum chemistry. Animals were sacrificedthe day after i.p. administration of occidiofungin. * Signifiesstatistically significant differences between treated and control group.B: Hematology WBC (1000/μl) 4.1 ± 0.7 5.0 ± 1.4 NEU (%) 18.1 ± 2.6  30.8± 10.8 LYM (%) 73.8 ± 4.0  62.5 ± 12.7 MONO (%) 5.4 ± 2.5 4.6 ± 2.0Serum Biochemistry ALP (U/L) 81.5 ± 17.3 75.6 ± 11.7 ALT (U/L) 100.0 ±44.6  20.4 ± 4.4  AST (U/L) 95.5 ± 37.1 69.4 ± 12.0 GGT (U/L) 8.0 ± 0.87.2 ± 2.3 CK (U/l) 154.0 ± 44.0  140.8 ± 30.4  BUN (mg/dL) 16.0 ± 0.8 13.6 ± 1.0 CREAT (mg/dL) 0.25 ± 0.05 0.20 ± 0.09 Effect of occidiofunginon hematology and serum chemistry. Animals were observed beforesacrificing on day 5 after subcutaneous administration of occidiofungin.

In the repeat-dose study, white blood cell differential counts showed anincrease in neutrophils and decrease in lymphocytes following a 5-day ipadministration of occidiofungin at 2 mg/kg (Table 5).

TABLE 5 Hematology and serum chemistry in 5 day repeat dose experiment.Single Dose of Occidiofungin 0 mg/kg/day 2 mg/kg/day Hematology WBC(K/ul) 3.5 ± 1.0 5.6 ± 0.4 NEU (%) 18.0 ± 3.3  45.9 ± 1.9* LYM (%) 75.8± 3.9  46.4 ± 0.9* MONO (%) 3.6 ± 0.7 4.8 ± 1.1 Serum Biochemistry ALP(U/l) 96.4 ± 9.5  39.6 ± 4.2* ALT (U/l) 49.8 ± 7.8  48.0 ± 6.5  AST(U/l) 104.0 ± 22.0  201.8 ± 59.6  GGT (U/l) 19.0 ± 6.0  15.2 ± 2.9  CK(U/l) 401.8 ± 123.9 401.0 ± 114.4 BUN (mg/dl) 24.4 ± 4.3  18.0 ± 2.8 CREAT (mg/dl) 0.38 ± 0.07 0.18 ± 0.09 Effect of occidiofungin onhematology and serum chemistry. Animals were observed before sacrificingon day 5 after i.p administration of occidiofungin. *Signifiesstatistically significant differences between treated and control group.

Although there were some statistically significant differences in serumclinical chemistry parameters, no consistent dose-response effects werenoted, which suggests that the significant effects represented normalbiological variation or experimental effects are not directly related tothe action of the test compound. Generally, no macroscopic findings wereobserved by histological examination. No observable differences werepresent in the microscopic cell morphology or macroscopic tissuemorphology of brain, liver, lung, thymus, or kidney. One finding was adecrease in activated thymocytes in the medulla in the 10 mg/kgsingle-dose toxicity study. This result in conjunction with decreasedthymus weight or increased neutrophil percentages suggests thatoccidiofungin causes a nonspecific stress response. Another likelyexplanation is that there is an allergic response to the presence of thexylose on occidiofungin causing an increase in neutrophils. In summary,there was no clear evidence for organ-specific histological effects ofoccidiofungin.

Intravenous (iv) administration of occidiofungin Six to eight-week-oldfemale BALB/c mice (5 mice per group) were given occidiofungin dissolvedin 1.5% hydroxy propyl-beta-cylcodextrin suspended in phosphate bufferedsaline (PBS). Spectrometric inspection at O.D.600 following addition ofvehicle to the purified dried drug had negligible absorbance differenceto vehicle without drug, suggesting that the drug went into solution.For the experiments, occidiofungin was administered by intravenous(i.v.) injection into the tail vein at a single dose at 5 mg/kg of bodyweight. The excipient control in each experiment matched the vehicle.Body weight and clinical signs (movement, posture, skin lesions,appearance of fur indicating normal grooming, and behaviors) wererecorded following administration at one, four, eight, sixteen, andtwenty-four hours. Necropsies were performed at 24 hours followingadministration of occidiofungin. Blood and tissue samples from animalsdosed at 5 mg/kg of body weight with occidiofungin in 1.5% hydroxypropyl-beta-cylcodextrin suspended in PBS were taken 24 hours followingexcipient or drug administration. Mice were anesthetized withisofluorane. Blood was then taken from the retroorbital plexus or heartpuncture for serum biochemistry assays (alkaline phosphatase, alanineaminotransferase, aspartate aminotransferase, albumin, and blood ureanitrogen) and hematology (white blood cell count and white blood celldifferentiation). Body weight was measured immediately before treatmentand 24 hours later before the mice were fully anesthetized and fixed in10% neutral buffered formalin. Histological examination was performed ona portion of each organ by using routine paraffin embedding techniqueand staining with hematoxylin and eosin (H & E).

Results of intravenous (iv) administration of occidiofungin are asfollows. Following administration of occidiofungin intravenously, miceweight ranged between 0% to 21% body weight-loss 24 hours aftertreatment and had an average weight loss of 6.2%. Excipient treated micebody weight showed 8% to 13% body weight gain 24 hours after treatmentand had a 9.6% average increase in body weight. A consistent behavioralresponse was observed at 1 hour and to a lesser extent at 4 hours posti.v. administration, in which the mice were more lethargic thanexcipient treated mice and had ruffled fur. They were responsive totouch but would move slower than excipient treated mice. Treatment wasnot associated with more typical rodent behaviors associated with severepain (e.g., writhing, vocalization, or lack of spontaneous locomotion).No other behavioral signs were observed. Mice behavior appeared to benormal by 8, 16, and 24 hour post injection. Generally, no macroscopicfindings were observed by histological examination performed on tissuesremoved from the euthanized mice at 48 hours post injection. Noobservable differences were present in the microscopic cell morphologyor macroscopic tissue morphology of esophagus, stomach, small intestine,colon, liver, pancreas, spleen, kidneys, lungs, heart and brain. Albuminand blood urea nitrogen (BUN) tests were similar for occidiofungintreated and excipient treated mice (Table 6). These blood tests areindicative of normal kidney and liver function. In addition, alkalinephosphatase (ALP) tests were similar between drug and excipient treatedmice (Table 6). These results indicate normal liver and bone cellfunction. Normally aspartate amino transferase (AST) and alanineaminotransferase (ALT) tests are performed in combination with ALP toassess liver function. Elevated levels of AST and ALT do suggest heartor liver damage, but do not necessarily indicate severe organ damage.Generally, a ratio of AST to ALT less than one is indicative of liverdamage. The ratio in all treated mice was greater than one, suggestingthat the liver is not damaged. AST values ranged from 177 to 1528 (U/1)with a mean value of 765 (U/1) and the ALT values ranged from 144 to1273 (U/1) with a mean value of 521 (U/1) (Table 6). Given thevariability in AST and ALT levels in treated mice, only the ALT levelswere statistically significant. White blood cell (WBC) counts were notstatistically different between treated and untreated mice. Thissuggests that occidiofungin i.v. administration at 5 mg/kg had nocytotoxicological effect on blood cells or bone marrow. The absence ofelevated levels further suggests normal spleen function. The percentageof neutrophils was statistically different in drug and excipient treatedmice, while the percentages of lymphocytes was not statisticallydifferent (Table 6).

TABLE 6 Percent body weight change, serum chemistry and hematologyfollowing intravenous administration of drug and excipient control.Single IV Dose of Occidiofungin 5 mg/kg 0 mg/kg *Weight Change % −6.2 ±9.9   +9.6 ± 1.9   Serum Biochemistry Albumin (g/dl) 3.1 ± 0.6 3.6 ± 0.2BUN (mg/dl) 29.6 ± 5   24.5 ± 3.3  ALP (U/l) 99 ± 34 116 ± 17  AST(SGOT) U/l 765 ± 692 165 ± 81  *ALT (SGPT) U/l 521 ± 652 36 ± 10Hematology WBC estimate 4750 ± 1225 5830 ± 1550 *Neutrophils % 43 ± 1416 ± 5  Lymphocytes % 54 ± 15 83 ± 7  Platelet estimate (xK/ul) 39 ± 2158 ± 58 Animals were sacrificed 24 hours following i.v. administrationof occidiofungin. *Signifies statistically significant differencesbetween treated and control group. p < 0.05 was considered statisticallysignificant.

The increase in neutrophils is likely attributed to an allergic responsedue to the presence of the xylose on occidiofungin. There was nostatistical difference in platelet counts between drug and excipienttreated mice, suggesting that occidiofungin does not affect plateletproduction by the bone marrow or destroy circulating platelets. Theincrease in neutrophils was not observed in mice treated with OF-Δxyl(Table 7). In summary, there was no evidence for organ specifichistological effects of occidiofungin. There were no apparentundesirable effects observed in the serum clinical chemistry andhematology parameters that would preclude additional animal testing ofthe compound. However, the increase in neutrophils does suggest apossible allergic response that appears to be alleviated with theremoval of xylose.

TABLE 7 Hematology following intravenous administration of occidiofunginand OF-Δxyl drug. Single IV Dose of Occidiofungin or OF-Δxyl (5 mg/kg)Hematology *Neutrophils % 26 ± 8.4  5 ± 0.7 Lymphocytes % 72 ± 9.2 95 ±0.7

Treatment of Vulvovaginal Candidiasis

Current antifungal treatment options are plagued with rapidly increasingoccurrence of resistance, high degree of toxicity and a limited spectrumof activity. Novel antifungal agents with a unique target, widerspectrum of activity, and reduced toxicity to the host are desired. Thisexample characterizes occidiofungin produced by Burkholderia contaminansMS14. The cellular target of the occidiofungin was determined to beactin. Actin binding metabolites are generally characterized by theirability to inhibit polymerization or depolymerization of actinfilaments, which presumably accounts for their severe toxicity.Occidiofungin, instead, has a subtler effect on actin dynamics thattriggers apoptotic cell death. The efficacy of the antifungal isdemonstrated in treating a vulvovaginal yeast infection in a murinemodel.

Occidiofungin has a wide spectrum of activity against filamentous andnon-filamentous fungi and minimal toxicity in an animal system. Themechanism of action of occidiofungin differs from the primary mode ofaction of the three common classes of antifungals. Occidiofungin hasbeen observed to rapidly induce apoptosis in yeast cells at the minimalinhibitory concentrations. The primary cellular target of occidiofunginis actin. Actin-mediated cellular processes in yeast, such asendocytosis, nuclear segregation and hyphal formation, were alldisrupted following addition of subinhibitory concentrations ofoccidiofungin. Occidiofungin's binding to actin interferes with F-actinfilament cable stability and does not interfere with polymerization ordepolymerization of actin filaments. Occidiofungin binds to F-actin withan estimated dissociation constant (Kd) of 1000 nM and has a highsaturation of binding ratio of more than twenty molecules ofoccidiofungin to one actin monomer. Given the high binding ratio, it waspredicted that the microscopic Kd value (which captures the affinity ofone occidiofungin molecule binding to actin) is significantly lower thanthe 1000 nM dissociation constant.

In the repeat-dose toxicity study, the hematology tests revealed thatwhite blood cell differential counts showed an increase in neutrophilsand decrease in lymphocytes. Neutrophils play important roles in hostdefense against all classes of infectious agents and pathology ofvarious inflammatory conditions. The increase in neutrophils alsosuggests a possible inflammatory or mild stress response. A potentialreason for the observed response is the presence of a xylose moiety onthe novel amino acid 2 (NAA2) or the NAA2 residue itself (FIG. 1).Mammalian immune system can recognize N-glycans containingβ-(1,2)-xylose and α-(1,3)-fucose residues. Novel analogs at the NAA2position of occidiofungin were synthesized to test whether the xylose onNAA2 residue is responsible for the development inflammation response.

In addition, occidiofungin has sub-micromolar activity against Pythiumspecies which lacks ergosterol in the membrane and against Cryptococcusneoformans which is resistant to echinocandins. Preliminarytoxicological analyses of occidiofungin using a murine model indicatedthat it was well tolerated at concentrations of 10 to 20 mg/kg, but itdemonstrates a mild allergic or stress response. Blood chemistryanalyses and histopathology performed on multiple organs showed atransient non-specific stress response with no damage to organ tissues.These data suggest that occidiofungin is a promising candidate fordevelopment as a clinically useful antifungal agent.

Spectrum of activity of occidiofungin against clinically relevant fungiOccidiofungin causes cell death in fungi through a mechanism of actionthat is distinct from the clinically used classes of antifungals. Due toits unique mechanism of action, occidiofungin has sub-micromolaractivity against azole and echinocandin resistant strains of fungi.Strains of Candida albicans, Candida glabrata, and Candida parapsilosisthat were resistant to fluconazole and caspofungin were sensitive tooccidiofungin (Table 8). Non-albicans strains are believed to be theprimary cause of recurrent vulvovaginal candidiasis. Furthermore,strains of Candida parapsilosis and C. neoformans that were resistant totreatment with caspofungin were found to be susceptible to treatmentwith occidiofungin. Occidiofungin was also found to have a broaderspectrum of activity than clinically available antifungals and was foundto be active against Aspergillus, Mucor, Fusarium, and Rhizopus species.Several strains of the dermatophyte Trichophyton were found to also besusceptible to occidiofungin treatment, including azole and terbinafineresistant strains. A summary of the results, as reported in Table 8,indicate that occidiofungin has activity against filamentous andnon-filamentous fungi at sub-micromolar concentrations and has a broaderspectrum of activity compared to other clinically available antifungals.Furthermore, sensitivity of fungal strains resistant to azoles andechinocandin class of antifungals, support the notion that occidiofunginis functioning via a novel mechanism of action.

Occidiofungin has sub-micromolar activity against azole and echinocandinresistant strains of fungi. Susceptibility of multiple strains of eachspecies (Filamentous fungi: Aspergillus fumigatus, Aspergillus flavus,Fusarium sp. (including solani and oxysporum), Mucor sp., Rhizopus sp.,Trichophyton rubrum, and Trichophyton mentagrophytes; and Yeasts: C.albicans, C. glabrata, C. krusei, C. parapsilosis, C. tropicalis, andCryptococcus neoformans) to occidiofungin was tested and antibioticresistant strains were used when available (Table 8). Fluconazoleresistant C. albicans and C. glabrata were sensitive to occidiofungin.Inhibitory concentrations of occidiofungin to these isolates were ≤2μg/mL, while the MIC of fluconazole against these strains was ≥32 μg/mL.Rhizopus spp. and C. neoformans were more sensitive to occidiofunginthan fluconazole. Cryptococcus neoformans is insensitive toechinocandins, but is susceptible to occidiofungin at sub-micromolarconcentrations.

Minimum inhibitory concentration (MIC) susceptibility testing wasperformed according to the CLSI M27-A3 and M38-A2 standards for thesusceptibility testing of yeasts and filamentous fungi, respectively.Incubation temperature was at 35° C. and the inoculum size was0.5-2.5×10³ colony-forming units (CFU)/mL and 0.4-5×10⁴ conidia/mL foryeasts and filamentous fungi, respectively. Inoculum concentration fordermatophytes was 1-3×10³ conidia/mL. RPMI was used throughout as thegrowth medium and Cryptococcus strains were tested in YNB. OccidiofunginMICs were recorded at 50% and 100% growth inhibition after 24 and 48hours of incubation, with the exception of dermatophytes which wereincubated for 96 hours. Fluconazole MICs against Candida strains wererecorded at 50% inhibition after 24 hours and against Cryptococcusstrains after 72 hours. Voriconazole MICs were recorded at 100%inhibition after 24 hours for zygomycetes and after 48 hours forFusarium and Aspergillus strains. Voriconazole MICs were recorded at 80%inhibition after 96 hours of incubation for dermatophytes. S. cerevisiaedeletion mutants were obtained from the commercially available BY4741deletion library (Thermo Scientific). Susceptibility testing was carriedout on inoculum size of 0.5-1×10⁴ cells/ml in YPD media at 30° C. andMICs recorded after 48 and 60 hours.

Development of Occidiofungin as a Treatment for VVC

The work that is being conducted will lead to a new, first-in-classanti-infective chemotherapeutic targeted specifically for fungalinfections. Fungal pathogens infect more than 7,000,000 adults in the USeach year and are a significant source of economic health cost forsociety. The lead molecule occidiofungin serves as a platform fordevelopment of t therapies to treat VVC.

In the United States it is estimated that seventy percent of the femalepopulation will suffer from VVC. A subset of this population, nearly5,000,000 women annually, suffer from RVVC. RVVC's estimated health carecost in the USA is $4-5 billion per year. This estimate is based onincreased medical costs to treat VVC and the economic impact from lostproductivity. The global cost of VVC and RVVC is expected to be over 10billion per year. VVC and RVVC also place a significant cost on qualityof life for any infected individual, their family, and employers.Although there are several preventative measures in development, thereis no effective therapeutic. An effective therapeutic that could treatVVC in a relatively non-invasive methodology could significantly reducethe cost to patients, while at the same time increase their quality oflife. Overall, a new therapeutic would reduce the impact of theseinfections on the economy. Additionally, there is a growing concern overthe increase in drug resistant strains of Candida and the lack of newtreatment of options available. These resistant strains are expected toincrease the global costs associated with RVVC.

Occidiofungin is superior in clinical setting as an antifungal forseveral reasons. For example, its antifungal activity has beendemonstrated against a wide array of fungi, including those resistant tocurrent antifungals in use. MICs of occidiofungin against Candidaspecies are between 0.5 and 2.0 μg/mL, which is similar in activity toechinocandins and amphotericin B. Pharmacodynamic (Time Kill)experiments revealed that occidiofungin is rapidly fungicidal againstCandida albicans, which is better than current clinically approvedantifungals. Occidiofungin retains its in vitro potency in the presenceof 5% and 50% human serum with a minimum lethal concentration (MLC) of 2and 4 μg/mL, respectively. An alternative target for occidiofungin otherthan targeting ergosterol production, binding to ergosterol, orinhibiting the 1,3-β-glucan synthase enzyme, is advantageous becausethese mechanisms are prone to resistance. In vivo toxicity studies showthat body and organ weight changes induced by occidiofungin are notassociated with any negative gross or microscopic findings. Hematologyand serum biochemistry tests reveal that occidiofungin does notsignificantly alter functions of organs. Occidiofungin demonstrates asignificant reduction in fungal load in murine VVC and system yeastinfection models.

These factors provide occidiofungin with unique advantages that arenecessary to be an effective treatment for VVC and RVVC.

Efficacy of Occidiofungin in Treating a Murine Model of VVC

Six to eight-week-old BALB/c mice were intravaginally infected with C.albicans and dosed once per day with occidiofungin for three days. Theoccidiofungin treated groups were compared to a vehicle control group.Three groups of six mice were treated with 100, 50, and 0 μg ofoccidiofungin suspended in 0.3% Noble agar. The occidiofungin treatedgroups reduced fungal load by more than two logs (FIG. 7). The reductionin fungal load with both treatment groups was statistically significantfrom vehicle control (p<0.001). There was no statistically significantdifference between the treated groups (p=0.33), suggesting that thelower limit of occidiofungin dosing was not achieved in the experiment.During the course of the study, the mice were examined for outward signsof distress or irritation. No behavioral changes including sluggishness,stretching, or reluctance to consume food was observed. Furthermore, novaginal bleeding or swelling was observed following treatment.

The murine model of VVC has been reported by Yano et al. A variation ofthis method was followed. Three groups of six mice were used to evaluatetwo concentrations of occidiofungin (100 μg and 50 μg) and vehiclecontrol (0.3% Noble agar). Briefly, six to eight-week-old BALB/c micewere treated subcutaneously with 200 ng per mouse of β-Estradiol17-valerate three days prior to inoculation with C. albicans (D-3). Asubcutaneous dose of estradiol was administered every three days (D0,D3) until the end of the experiment to induce pseudo-estrus.Approximately a 20 μL intravaginal inoculation of a 2.5×10⁶ colonyforming units (CFU)/mL of C. albicans defines day zero (D0) of the VVCstudy. On the same day of inoculation (D0), another subcutaneousinjection of estradiol was made. Lyophilized powder of occidiofungincontaining either 100 μg or 50 μg of occidiofungin was suspended in 20μL of warm 0.3% Noble agar before intravaginal inoculation. Drugtreatment was done on day 2 (D2), day 3 (D3) and day 4 (D4) of thestudy. On day 5 (D5), the vaginal lumen was lavaged with 100 μL ofsterile PBS with a 200 μL pipette tip. Serial dilutions and total colonyforming units per vaginal lavage were determined by plating on YPDplates containing 50 μg/mL of chloramphenicol. The colony forming units(CFUs) obtained from each lavage were counted on plates containing30-300 colonies for determining the CFU/mL estimates. Body weight, signsof vaginal irritation such as swelling or bleeding and clinical signs ofdiscomfort (stereotypical stretching behavior) were monitored.Statistical analyses (T-test) were done to compare the control group totreated groups and to compare differences between treated groups. Allthe analyses were 2-sided, with P<0.05 considered statisticallysignificant.

TABLE 8 Activity of occidiofungin against filamentous andnon-filamentous fungi. Voricona- Flucona- Occidiofungin (μg/mL) zolezole 24 hours 48 hours 72 hours 96 hours MIC MIC Species 50% 100% 50%100% 50% 100% 80% 100% (μg/mL) (μg/mL) *Trichophyton 1 2 0.25 >16mentagrophytes 10207 Trichophyton 1 2 0.06 >16 mentagrophytes 28556Trichophyton 1 2 0.06 16 mentagrophytes 28641 ^(&) Trichophyton 1 20.008 0.25 rubrum 11199 Trichophyton 1 2 0.03 2 rubrum 28658Trichophyton 1 2 0.03 2 rubrum 28659 Rhizopus 4 8 — 8 16 microsporus28506 Rhizopus 4 8 — 8 >16 oryzae 28403 Rhizopus 2 4 — 8 >16 microsporus27785 Mucor 4 8 4 8 >16 circinelloides 19445 Mucor 2 4 — 4 >16 racemosus27784 Mucor 2 4 — 4 >16 fragdis 27782 Fusarium 2 4 — 4 >16 solani 28386Fusarium 2 4 — 4 >16 oxysporum 27718 Fusarium 2 4 2 4 >16 solani 18749Aspergillus — 4 — 4 1 flavus 28517 Aspergillus 2 4 — 4 2 flavus 28455Aspergillus 2 4 — 4 2 flavus 28445 Aspergillus — 4 — 4 1 fumigatus 28434Aspergillus — 2 — 2 1 fumigatus 28435 Aspergillus 2 4 2 4 1 fumigatus28436 ^(#) Candida — 1 — 2 32 albicans 23512 Candida 4 8 4 8 8 albicans28200 Candida — 2 — 2 0.125 albicans 28102 ^(#) Candida 2 4 — 4 64glabrata 27243 Candida — 2 — 2 4 glabrata 25742 Candida 4 8 4 8 >64glabrata 28271 Candida 2 4 — 4 16 krusei 9541 ^(#) Candida 4 8 4 8 64krusei 28415 Candida 4 8 4 8 16 krusei 28570 ⁺ Candida 2 4 — 4 0.125parapsilosis 2006 Candida 4 8 4 8 0.25 parapsilosis 28364 Candida — 4 —4 0.25 parapsilosis 28174 Candida — 2 — 2 0.25 tropicalis 9624 Candida 48 4 8 0.125 tropicalis 28272 Candida 4 8 4 8 0.125 tropicalis 28478 ⁺Cryptococcus — 2 4 neoformans 19526 Cryptococcus — 2 2 neoformans 27708Cryptococcus — 1 4 neoformans 28446

Example 6—Actin Binding Properties of Occidiofungin Analogs

To characterize the actin binding properties of the dodecylamine anddihydrosphingosine analogs of occidiofungin, a co-sedimentation assaywas performed. Native occidiofungin had a Kd value of 1000 nM and asaturation of binding ratio of ˜25:1 (ligand:actin monomer, FIG. 9A).Further, phalloidin had an estimated Kd value of ˜8 nM with a saturationof binding ratio of ˜0.7:1 (ligand:actin monomer, FIG. 9C). Thedodecylamine analog was determined to have a Kd value of 4200 nM with asaturation of binding ratio of ˜34:1 (ligand:actin monomer) (FIG. 9B).Interestingly, the dihydrosphingosine analog had a Kd value of 25 nMwith a saturation of binding ratio of ˜1.8:1 (ligand:actin monomer, FIG.9D). The dodecylamine analog had a similar saturation of binding toactin as the native compound with approximately a 4-fold lower bindingaffinity to actin. The dihydrosphingosine analog showed an actin bindingproperty closer to phalloidin's and had a 13.5-fold lower saturation ofbinding ratio than native occidiofungin. The decrease in dissociationconstant is likely attributed to the low saturation of binding for thedihydrosphingosine analog compared to native occidiofungin and not anincrease in affinity to actin.

Given that the actin binding properties of the dihydrosphingosine analogwere different from native occidiofungin, the analog was tested todetermine whether its mechanism of actin binding was different thannative occidiofungin. Native occidiofungin does not interfere with actinpolymerization or depolymerization, but rather interferes with actincable formation triggering ROS accumulation and apoptotic cell death.The molar ratio of occidiofungin, analogs of occidiofungin, orphalloidin to actin in the polymerization and depolymerization assay wasclose to 1:1. The pyrene fluorescence readings approximately one hourafter incubation in the polymerization buffer plateaued at around 19,000for phalloidin and 21,000 AU for the dihydrosphingosine analog andnative occidiofungin (FIG. 10). Following incubation in thedepolymerization buffer, actin filaments incubated with phalloidin hadpyrene fluorescent readings above 5,000 AU, while actin filamentsincubated with dihydrosphingosine analog and native occidiofungin hadsimilar readings as no drug control at 2,500 AU. Phalloidin completelyblocked the depolymerization of F-actin. The dihydrosphingosine analogdid not interfere with polymerization or depolymerization of actin,similar to what was observed with native compound (FIG. 10). The lack ofany interference with the polymerization or depolymerization of actin bythe dihydrosphingosine analog supports the assumption that thedihydrosphingosine analog and native occidiofungin have the same bindingregion on actin.

Example 7—Improved Analogs of Occidiofungin

This example provides strategies to produce novel analogs ofoccidiofungin. Oxidative cleavage of the diol group present in thealiphatic chain of NAA2 followed by reductive aminations of alkyl aminesrestored the aliphatic structure of the native compound. The methodsdescribed to synthesize the analogs of occidiofungin provide anefficient strategy for generating novel analogs of occidiofungin. One ofthe analogs synthesized was identified to have reduced antifungalactivity but significantly lower dissociation constant and stoichiometryin an actin co-sedimentation study. These results showed that the fattyacid moiety in native occidiofungin plays an important role with regardsto its antifungal activity and actin binding properties.

Occidiofungin needs to enter the cell to reach its cellular targetactin. Against S. cerevisiae, the undecylamine and dodecylamine analogswere both inactive at a concentration that was sixty-four-fold higherthan native compound, while the dodecylamine analog only had a four-foldreduction in affinity for actin. The lack of activity of undecylamineand dodecylamine analogs suggests that the hydroxyl groups on the sidechain may be essential for the uptake of occidiofungin into susceptiblecells. This is supported by the fact that the dodecylamaine analog wascapable of binding to actin in a similar manner as native occidiofungin.Given that the inhibitory activity of the dihydrosphingosine analog canbe partially restored, the introduction of the hydroxyl groups near thebase of the lipid moiety may aid in cellular entry and enable the analogto reach the actin target.

Accordingly, some embodiments of the invention provide modifying anoccidiofungin analog disclosed herein to render it suitable for entryinto the cell. Such modification can be attachment, for example, via acovalent bond, to a moiety that facilitates entry of the occidiofunginanalog into a cell. Such modification can also be inclusion of theoccidiofungin analogs disclosed herein into vesicles or capsules thatfacilitate uptake of the occidionfungin analog into a cell. The vesiclesor capsules can be lipid vesicles or1-Palmitoyl-2-oleoylphosphatidylcholine (POPC) vesicles.

Intravenous administration of occidiofungin triggered a mildinflammation or allergic response. Xylose is only reported to be presentin plants and some invertebrates and is not known to be present inmammals. β-(1,2)-xylose is involved in a common cross-reactive antigenicdeterminant (CCD), and is shared by a variety of glycoproteinsrecognized by IgE antibodies of patients with food or respiratoryallergies. Both IgE and IgG2 antibodies responded to CCDs and that CCDsaffected both Th1- and Th2-type responses. β-(1,2)-xylose modificationsof N-glycans modify specific IgE-binding in individuals allergic to Lyce 2, a glycosylated allergen of tomato. The xylose free analogs ofoccidiofungin disclosed herein provide therapeutically superior analogsof occidiofungin.

The dihydrosphingosine analog exhibits low binding constant andsaturation stoichiometry. The high saturation of occidiofungin to actin(25:1) can be attributed to the self-assembly mechanism of nativeoccidiofungin monomers following its binding to F-actin. This is notuncommon activity for lipopeptides and the formation of self-assembledcomplexes was observed with several lipopeptide antibiotics. Thepolarity of dihydrosphingosine analog is much less than the nativecompound (eluted at 33% versus 47% water on HPLC; FIGS. 5-6), whichmight contribute to the loss of the self-assembly mechanism, in thesense that amphiphilicity leads to self-assembly at high concentrationsfor peptide amphiphiles. However, the relationship between self-assemblyand bioactivity is complicated and remains unclear. Reduced antifungalactivity of the dihydrosphingosine analog may be attributed to thereduced polarity and self-assembly property. The dihydrosphingosineanalog had a Kd value of 25 nM and had a stoichiometry closer to a 1:1(ligand:actin) binding. The dihydrosphingosine analog may have a higheraffinity for actin. The lower Kd value likely represents the microscopicdissociation constant (which captures the affinity of one occidiofunginbinding to actin) versus the macroscopic Kd value captured by the nativecompound (24:1 ligand:actin).

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims. In addition, anyelements or limitations of any invention or embodiment thereof disclosedherein can be combined with any and/or all other elements or limitations(individually or in any combination) or any other invention orembodiment thereof disclosed herein, and all such combinations arecontemplated within the scope of the invention without limitationthereto.

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1-26. (canceled)
 27. An analogue of an occidiofungin or salt thereof,the occidiofungin having Formula I:

wherein the analog or salt thereof does not contain xylose moiety fromFormula I (OF-Δxyl analogue).
 28. The OF-Δxyl analogue or salt thereofclaim 27, having Formula II:


29. The OF-Δxyl analogue or salt thereof claim 27, having Formula III:


30. The OF-Δxyl analogue or salt thereof of claim 29, wherein R₄ or R₅of Formula III is independently H, OH, a substituted or unsubstitutedalkane, substituted or unsubstituted alkene, substituted orunsubstituted alkyne, substituted or unsubstituted aryl, substituted orunsubstituted heterocycle, substituted or unsubstituted heteroaryl,substituted or unsubstituted heterocycle, substituted or unsubstitutedether, substituted or unsubstituted thioether, substituted orunsubstituted ketone or a halogen.
 31. The OF-Δxyl analogue or saltthereof of claim 27, having Formula IV:


32. The OF-Δxyl analogue or salt thereof of claim 27, having Formula V:


33. The OF-Δxyl analogue or salt thereof of claim 32, wherein R₆ ofFormula V is phenyl, 4-nitro phenyl, 2-nitrophenyl or 2,4-dinitrophenyl.34. The OF-Δxyl analogue or salt thereof of claim 33, wherein R₆ is2-aminophenyl, 4-aminophenyl or 2,4-diaminophenyl, wherein one or moreamino group(s) is/are optionally substituted.
 35. The OF-Δxyl analogueor salt thereof of claim 34, wherein the one or more amine group(s) of2-aminophenyl, 4-aminophenyl or 2,4-diaminophenyl is/are substitutedwith OH, a substituted or unsubstituted alkane, substituted orunsubstituted alkene, substituted or unsubstituted alkyne, substitutedor unsubstituted aryl, substituted or unsubstituted heterocycle,substituted or unsubstituted heteroaryl, substituted or unsubstitutedheterocycle, substituted or unsubstituted ether, substituted orunsubstituted thioether, substituted or unsubstituted ketone or ahalogen.
 36. A pharmaceutical composition comprising an OF-Δxyl analogueor salt thereof of claim 27 and a pharmaceutically acceptable vehicle.37. A method of treating or preventing a fungal infection, comprisingadministering to a subject in need thereof an effective amount of anOF-Δxyl analogue or salt thereof of claim 27 or a pharmaceuticalcomposition thereof.
 38. The method of claim 37, comprisingadministering the OF-Δxyl analogue or salt thereof or the pharmaceuticalcomposition intravaginally, intramuscularly, subcutaneously,intrathecally, intravenously or intraperitoneally.
 39. The method ofclaim 37, wherein the fungal infection is caused by a Candida spp.,Trichophyton spp., Rhizopus spp., Mucor spp., Fusarium spp., Aspergillusspp. or Cryptococcus spp.
 40. The method of claim 39, wherein theCandida spp. is C. albicans, C. glabrata, C. krusei, C. krusei, C.parapsilosis or C. tropicalis.
 41. A method of producing an OF-Δxylanalogue of Formula II or salt thereof, comprising an oxidative cleavagebetween the fifth and sixth carbons of NAA of an occidiofungin havingFormula I, wherein the oxidative cleavage is designed to convert thefifth carbon to a carbonyl of a resulting aldehyde.
 42. The method ofclaim 41, wherein the occidiofungin of Formula I is treated withmetaperiodic acid (HIO₄), sodium periodate (NaIO₄) or potassiumperiodate (KIO₄) for about 20-40 minutes at about 60-80° C.
 43. A methodof producing an OF-Δxyl analogue of Formula III or salt thereof,comprising a first step of treating the OF-Δxyl analogue of Formula IIwith a primary or secondary amine in the presence of an alcohol toproduce an imine containing OF-Δxyl analogue and a second step ofreducing the imine containing OF-Δxyl analogue with a reducing agent toproduce the OF-Δxyl analogue of Formula III.
 44. The method of claim 43,wherein the alcohol is methanol, the reducing agent is NaBH₄, and theprimary or secondary amine is undecylamine, dodecylamine orDL-dihydrosphingosine.
 45. A method of producing an OF-Δxyl analogue ofFormula IV or salt thereof, comprising reacting an OF-Δxyl analogue ofFormula II with a substituted or unsubstituted triazole.
 46. The methodof claim 45, comprising a first step of treating the OF-Δxyl analogue ofFormula II with the substituted or unsubstituted triazole in thepresence of an alcohol to produce an intermediate OF-Δxyl analogue and asecond step of reducing the intermediate OF-Δxyl analogue with areducing agent to produce the OF-Δxyl analogue of Formula IV.
 47. Themethod of claim 46, wherein the alcohol is methanol and the reducingagent is NaBH₄.
 48. A method of producing an OF-Δxyl analogue of FormulaV or salt thereof, comprising reacting an OF-Δxyl analogue of Formula IIwith a hydrazine.
 49. The method of claim 48, comprising a first step oftreating the OF-Δxyl analogue of Formula II with a hydrazine in thepresence of an alcohol to produce an intermediate OF-Δxyl analogue and asecond step of reducing the intermediate OF-Δxyl analogue with areducing agent to produce the OF-Δxyl analogue of Formula V.
 50. Themethod of claim 49, wherein the alcohol is methanol and the reducingagent is NaBH₄.
 51. The method of claim 48, wherein the hydrazine isphenyl hydrazine, 2-nitrophenyl hydrazine, 4-nitrophenyl hydrazine or2,4-dinitrophenyl hydrazine.
 52. The method of claim 51, furthercomprising reducing one or more nitro-groups from 2-nitrophenylhydrazine, 4-nitrophenyl hydrazine or 2,4-dinitrophenyl hydrazine toamines.